KLONING DAN EKSPRESI PROTEIN P Domain TERINTEGRASI EPITOP HBsAg DAN EPITOP HBcAg (Chimeric P Domain-VHB) MENGGUNAKAN SISTEM PROTEIN FUSI Glutathione S-transferase (GST) PADA Escherichia coli BL21 (DE3)

• Main Author: Handoko, Claireta
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/42767
• Institution: Institut Teknologi Bandung

P Particle is a potential vaccine platform for developing intranasal vaccine. This particle composed of 24 monomers of P Domain which derived from Norovirus structural capsid protein. The production of P Domain can be performed using Escherichia coli expression system. However, heterologous expression using prokaryote system can cause recombinant protein expressed as inclusion bodies. One of the solutions to overcome this problem is using fusion protein system that can stabilize the hydrophilic site of the protein during the folding process and cause the protein expressed in a soluble form. Glutathione S-transferase (GST) is a fusion protein system in pGEX series vector commonly used in P Domain production. In this research, gene which codes P Domain from Norovirus GII.4 integrated with determinant ‘a’ epitop of HBsAg and 18-27 epitop of HBcAg (Chimeric P Domain-VHB) is used. Chimeric P Domain-VHB is a potential antigen for intranasal vaccine against Hepatitis B and Norovirus. The purposes of this research are cloning of gene encoding Chimeric P Domain-VHB in pGEX18, analyze the expression of Chimeric P Domain-VHB fused with GST in E. coli BL21(DE3), and optimization of IPTG concentration to improve the solubility of Chimeric P Domain-VHB fused with GST. Cloning of gene of interest in pGEX18 was conducted with restriction and ligation process. The cloning result (pGEX_PDomain-VHB) was confirmed with colony PCR, plasmid isolation, restriction, and sequencing. The confirmed pGEX_PDomain-VHB was used for the expression of Chimeric P Domain-VHB fused with GST in E. coli BL21(DE3) using Isopropyl ?-D-1-thiogalactopyranoside (IPTG) as the inducer. Profile analysis of total protein, insoluble protein fraction, and soluble protein fraction were performed using SDS PAGE. Optimization of IPTG concentration within 4 hours of induction was conducted to improve the percentage of soluble protein of interest. Percentage of soluble Chimeric P Domain-VHB fused with GST was generated from semiquantitative analysis using ImageJ software. Based on the result, cloning process was successfully conducted and resulted pGEX_PDomain-VHB as the output. It is suspected that Chimeric P Domain-VHB fused with GST has been successfully expressed in E. coli BL21(DE3). This suspicion was based on the appearance of 70.5kDa protein band in SDS PAGE result. However, according to the one-way ANOVA statistical test, the variation of IPTG concentrations in 4 hours of induction does not give a significant result to the percentage of soluble Chimeric P Domain-VHB fused with GST.